Chicken Collagen Hydrolysate Protects Rats from Hypertension and Cardiovascular Damage

Abstract

We previously reported that chicken collagen hydrolysate (CCH) has strong angiotensin I converting enzyme (ACE) inhibitory activity and antihypertensive effects on spontaneously hypertensive rats. Here, we investigated the chronic therapy effects of CCH on blood pressure and vascular relaxation in a cardiovascular damage model of Wistar-Kyoto rats induced by N-nitro-l-arginine methyl ester (l-NAME). Following co-treatment with CCH for 4 weeks, the increment of systolic blood pressure was suppressed significantly. At 8 weeks, the vasorelaxation of thoracic aorta increased significantly, and cardiovascular damage was ameliorated. The concentration of soluble intercellular adhesion molecule-1 (ICAM-1) in blood was reduced significantly by long-term administration of CCH, whereas the nitric oxide concentration was increased significantly at 1 hour post-treatment. The results suggest that beneficial effects of CCH result from antihypertensive function, but also from inhibition of cardiovascular damage to the endothelial cells via its ACE inhibitory activity and regulation of nitric oxide and ICAM-1, which suggests that CCH may be useful as a medicinal food for patients with cardiovascular disease.

Introduction

H ypertension is a major risk factor for coronary heart disease, stroke, congestive heart failure, renal insufficiency, and peripheral vascular disease.¹ Prevention of hypertension is therefore very important. Recently, ingredients derived from foods, including dried bonito,² sardines,³ skipjack tuna,⁴ wakame seaweed,⁵ and soybean,⁶ have been shown to have antihypertensive effects, and some of these materials are now marketed as functional food products for people with hypertension.

Chicken extract is widely used as a food throughout the world. Chicken broth contains abundant nitrogenous compounds such as free amino acids and peptides, as well as inosinic acid.⁷ Collagen is a major component of the extracellular matrix and exists as a main structural protein for animals, including mammals, birds, and fish. The effects of collagen on suppressing increases in blood viscosity during shock, improving the microcirculation, and promoting recovery and stabilization of blood pressure have been reported.⁸ However, the basic mechanism of these effects has not been fully clarified.

Previously, we have reported that chicken breast muscle hydrolysate exerts an antihypertensive effect in spontaneously hypertensive rats.⁹ Collagen-derived angiotensin I converting enzyme (ACE) inhibitory peptides have been isolated as the active entity.^10,11 The antihypertensive effect of chicken collagen hydrolysate (CCH) has also been confirmed in mildly hypertensive subjects. We deduced that, in addition to its antihypertensive effect, the CCH had a protective effect on the blood vessels through the activation of endothelial progenitor cells.¹²

ACE converts angiotensin I to angiotensin II, which is a strong vasopressor, and inactivates bradykinin, a vasodilator.¹³ Studies in animals show that chronic administration of N-nitro-l-arginine methyl ester (l-NAME) increases ACE activity in cardiovascular tissues, and co-treatment with ACE inhibitors or AT1 receptor antagonists prevents l-NAME-induced arteriosclerotic changes.^14,15 Administration of l-NAME blocks the synthesis of nitric oxide (NO) and results in vascular structural changes, hypertrophy, and fibrosis of the myocardium in animals.^16,17

Endothelial NO inhibits platelet aggregation, thrombogenesis, leukocyte adhesion, and proliferation of vascular smooth muscle cells.^18,19 Decrement of NO from the vascular beds results in increased leukocyte–endothelium interaction via up-regulation of endothelial cell adhesion molecules, which include intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1.²⁰ Under this condition, numerous leukocytes adhere to the vascular endothelium and migrate across it, thus aggravating endothelial dysfunction and tissue injury.²¹ In contrast, systemic administration of NO donors to NO-deficient animals preserves endothelial function and attenuates pathological interactions between circulating leukocytes and the vascular endothelium.

CCH, with its antihypertensive effect, may be useful as a functional food in the treatment of cardiovascular diseases such as hypertension and atherosclerosis, but this issue has not yet been fully explored.

In this study, we investigated whether CCH can prevent hypertension and cardiovascular disorders, as well as the pathway of the putative protective effect of CCH on structural cardiovascular remodeling in a model of cardiovascular damage induced by l-NAME in Wister-Kyoto (WKY) rats.

Materials and Methods

CCH preparation

The unfeathered portions of chicken legs (with claws) were solubilized by acid treatment and then digested with Aspergillus oryzae protease (Mitsubishi-Kagaku Food Co., Tokyo, Japan). Fractions with molecular masses less than 3,000 Da were collected with an ultrafiltration membrane (Millipore Co., Bedford, MA, USA) and were designated as CCH. After ultrafiltration, CCH solution was eluted on an activated carbon column for decolorization and then lyophilized.

Experimental animals

Eight-week-old male WKY rats were fed on a commercial chow (MF; Oriental Yeast, Tokyo) and water for 2 weeks ad libitum in an environmentally controlled room (23°C, 55% humidity). Then, 36 male WKY rats were randomly divided into three groups. The first group (control group) received untreated chow and drinking water. The second group (l-NAME group) received l-NAME in the drinking water (0.5 g/L) for 8 weeks (l-NAME [1.0 g/L] for the additional experiment). The third group (L + CCH group) received l-NAME in the drinking water and CCH (2.0 g/kg/day) by a metal oral Zonde needle. All animal procedures were carried out in accordance with the “Animal Experimentation Guidelines of the Japanese Association for Laboratory Animal Science” and were approved by the Animal Use and Care Committee of Nippon Meat Packers, Inc. (Tsukuba, Ibaraki, Japan).

Measurement of systolic blood pressure (SBP)

Tail SBP was measured every 2 weeks by the tail-cuff method with a plethysmographic tail apparatus (model 98A, Softron Co., Tokyo). Before the measurements, the rats were kept at 37°C for 10 minutes to make the pulsations of the tail artery detectable.

Vasorelaxation assay

The vasorelaxation assay was performed on eight or nine rats from each group at 8 weeks post-treatment. The rats were euthanized with diethyl ether, and the thoracic aorta was removed and placed in Krebs-Henseleit solution at 4°C.²² The solution contained the following chemicals: 118.3 mM NaCl, 4.7 mM KCl, 2.0 mM CaCl₂, 1.2 mM MgSO₄, 25.0 mM NaHCO₃, 1.2 mM KH₂PO₄, 0.026 mM calcium EDTA, and 11.1 mM glucose. The surrounding connective tissue and fat were carefully removed away from the thoracic aorta and cut into 2–3-mm-wide rings. Segments of thoracic aorta were mounted between two steel hooks in isolated tissue chambers containing Krebs-Henseleit solution (pH 7.4) at 37°C; the solution was continuously aerated with a mixture of 95% O₂ and 5% CO₂. An optimum resting tension of 1.0 g was applied to all aortic segments, and the tension was adjusted every 15 minutes during a 45-minute equilibration period before addition of the reagents. The isometric tension was recorded with an isometric force displacement transducer (model UL-10GR, Mineber, Tokyo) connected to an acquisition system (MSC-2, Labo Support Co., Ltd., Osaka, Japan). After the equilibration period, segments were initially exposed twice to 75 mM KCl to test their functionality and their maximum contractility. After a 60-minute washout period, l-norepinephrine bitartrate (1 μM) was added to cause precontraction. This was followed by the addition of cumulative doses of acetylcholine chloride to the bath solution to produce relaxation. Vascular relaxation was expressed as a percentage of tension development.

Determination of soluble ICAM-1 (sICAM-1) concentration

Plasma sICAM-1 levels were determined every 4 weeks by using commercially available enzyme-linked immunosorbent assay kits (R& D Systems Inc., Minneapolis, MN, USA).²³ Blood samples were collected from the tails of rats using a heparin-treated capillary tube and then centrifuged for 20 minutes at 2,000 g. Anti-sICAM-1 monoclonal antibody labeled with horseradish peroxidase (R& D Systems Inc.) and sICAM-1 standards and samples were added to the wells and incubated for 2 hours at room temperature. The wells were washed five times, and then the substrate solution was added. After 30 minutes of incubation at room temperature, the reaction was stopped by addition of 0.1% HCl. Absorbance was measured at a wavelength of 450 nm, with the correction wavelength set at 540 nm, on a model 3550 microplate reader (Bio-Rad, Tokyo).

Determination of serum NO_x concentration

Blood samples were collected from the tails of rats anesthetized with diethyl ether and centrifuged at 10,000 g for 5 minutes. The supernatant was mixed with an equal amount of ethanol and then centrifuged at 10,000 g for 5 minutes. The supernatant collected was used for measurement of NO_x, as previously described.²⁴ The sample was put into an automatic sample injector connected to an automatic detector–high-performance liquid chromatography system (model ENO-20, Eicom, Kyoto, Japan). NO₂ ⁻ and NO₃ ⁻ in the dialysate were separated on a reverse-phase separation column packed with polystyrene polymer (NO-PAK, 4.6–50 mm; Eicom), and NO₃ ⁻ was reduced to NO₂ ⁻ in a reduction column packed with copper-plated cadmium filings (NO-RED; Eicom). NO₂ ⁻ was mixed with a Griess reagent to form a purple azo dye in the reaction coil. Contamination of NO₂ ⁻ and NO₃ ⁻ in Ringer's solution and the reliability of the reduction column were examined in each experiment.

Histopathological observations

Histopathological observation was performed by a single observer who was blinded to all treatment protocols. Five paraffin-embedded sections were prepared from each tissue, as previously described.²⁵ In brief, tissues isolated from each group were fixed with formalin, then embedded in paraffin, sectioned, and stained with hematoxylin and eosin or Masson's trichrome staining solution. Slices were examined under a light microscope for histopathological changes. Representative sections were photographed by an automatic photomicrographic system (Vanox, Olympus, Tokyo).

Statistical evaluations

All data were analyzed and expressed as mean ± SE values. Comparisons were performed by Tukey's test to detect differences between the groups. A probability value less than .05 was considered as statistically significant.

Results

Survival curves

At 8 weeks post-treatment, the survival rate of l-NAME group rats, which had received l-NAME (0.5 g/L) in the drinking water, was 66.7% of that of control rats. However, rats that had ingested CCH (L + CCH group) had a significantly improved survival rate (91.7% of the control) compared with that of the l-NAME group (P < .05) (Fig. 1). During all of the experiments, monitoring revealed that the rats drank 17–30 mL of water and ate 16–30 g of chow every day, confirming that their drinking and eating patterns were unaffected by the treatment protocols. Body weight gains did not differ among groups (data not shown).

FIG. 1.

Survival curves of the control group, l-NAME group, and L + CCH group during 8 weeks of treatment. Data are mean ± SE values (n = 12 rats). *P < .05 versus l-NAME group.

Effect on antihypertensive measures

The effects of oral administration of l-NAME and l-NAME + CCH for 8 weeks on SBP are shown in Figure 2. Baseline SBP was similar among all the groups. Unlike the control group, SBP in the l-NAME group and the L + CCH group increased slowly in a time-dependent manner. The rate of increase for SBP in the L + CCH group was decreased from 1 week post-treatment. At week 4 post-treatment, SBP in the L + CCH group was significantly lower than that of in the l-NAME group, and the absolute value was approximately 20 mm Hg.

FIG. 2.

Changes of SBP in the control group, l-NAME group, and L + CCH group during 8 weeks of treatment. Tail SBP was measured by a tail-cuff method every 2 weeks. Results were expressed as changes from baseline SBP measured 1 week before treatment with l-NAME. Data are mean ± SE values (n = 8–12 rats). *P < .05 versus l-NAME group.

Effect on vasorelaxant measures

Figure 3 shows the relaxation caused by administration of CCH for 8 weeks, as determined by screening of the rat thoracic aortic segments. Treatment with acetylcholine chloride caused concentration-dependent relaxation of the thoracic aortic preparations from all groups after the preparations had been precontracted with L-norepinephrine bitartrate. The acetylcholine chloride induced a relaxant response in the thoracic aorta from the l-NAME group (12.7 ± 4.9% vasorelaxation), which was significantly less than that in preparations from the control group (69.5 ±8.1%). Compared with that of the l-NAME group, vasorelaxation of thoracic aorta from the L + CCH group (36.0 ± 6.1%) was significantly improved by chronic administration of CCH (P < .05).

FIG. 3.

Relaxation of rat thoracic aortas. Rat thoracic aorta was precontracted with l-norepinephrine bitartrate (1 μM), and acetylcholine chloride (ACh) was applied cumulatively to the bath solution. Vascular relaxation was expressed as a percentage of tension development before application of ACh. Data are mean ± SE values (n = 8–9 rats). *P < .05 versus l-NAME group.

Effects on plasma sICAM-1 concentration

Plasma sICAM-1 levels did not differ significantly between 4 and 8 weeks post-treatment with l-NAME (4 weeks, 15.03 ± 0.65 ng/mL; 8 weeks, 15.14 ± 0.40 ng/mL) (Fig. 4). Furthermore, there was also no significant change in plasma sICAM-1 level in the L + CCH group between 4 and 8 weeks post-treatment (4 weeks, 13.91 ± 0.34 ng/mL; 8 weeks, 14.23 ± 0.36 ng/mL). However, the plasma sICAM-1 in the L-CCH group rats was significantly lower than that in the l-NAME group at both 4 and 8 weeks post-treatment (P < .05).

FIG. 4.

Concentration of sICAM-1 in rat plasma. Blood samples were collected from the tail vein into heparinized capillary tubes and then centrifuged at 12,000 g for 5 minutes at 4°C. Plasma was collected and stored at −80°C until analysis. Data are mean ± SE values (n = 8–9 rats). *P < .05 versus l-NAME group.

Changes in serum NO_x concentration

Changes in NO_x were measured in the sera of the rats. After 8 weeks, serum NO_x concentrations did not differ significantly among the groups (data not shown). However, we found in an additional experiment that the NO_x concentration in the serum of rats that ingested CCH was significantly greater than that of control rats that ingested water at 1 hour post-treatment, whereas at 2 hours post-treatment they were nearly same (Fig. 5). This result indicated that CCH treatment could increase the NO concentration transiently.

FIG. 5.

Transient change of NO_x concentration in serum from a rat treated with a single dose of CCH (2 g/kg of body weight). Blood samples were collected from the tail vein of the rat, stored at 4°C for 10 minutes, and then centrifuged at 10,000 g for 5 minutes at 4°C. Serum was collected and stored at −80°C until analysis. Data are mean ± SE values (n = 6 rats). *P < .05 versus no CCH treatment.

Histopathology of heart, kidney, and thoracic aorta

The size of focal lesions and the extent of invasion by inflammatory cells were evaluated by histopathological methods. The results revealed that the focal fibrosis appeared in sections of rat heart from the l-NAME group stained with hematoxylin and eosin or Masson's trichrome, whereas the mild fibrosis was present in the control group and L + CCH groups (Fig. 6A). In the kidney sections from an l-NAME group rat, glomerulosclerosis and focal fibrosis were moderate, and the cortex showed omission or denaturation of nephrons. These findings were not present in the control group and the L + CCH group (Fig. 6B). No changes were found in the thoracic aorta in any of the groups (Fig. 6C).

FIG. 6.

Histopathological photomicrographs of sections from the control group, l-NAME group, and L + CCH group rats at 8 weeks post-treatment: (A) heart sections stained with hematoxylin and eosin (HE) and Masson's trichrome (MT), (B) renal sections stained with HE stain, and (C) thoracic aorta sections stained with MT stain. Bar = 100 μm.

Discussion

This study demonstrated that treatment with CCH partially restored survival rates and attenuated the increase in inflammatory and proliferative changes induced in the heart, kidney, and thoracic aorta by l-NAME treatment. We also confirmed that chronic administration of l-NAME caused systemic arterial hypertension in the rat in vivo.^16,26

We previously reported that CCH reduced blood pressure in spontaneously hypertensive rats by inhibiting ACE activity.^9,11 Several reports have shown that local expression of ACE plays a key role in the pathogenesis of the coronary vascular and myocardial remodeling induced in rats by long-term administration of l-NAME.¹⁴ Our result showed that CCH treatment reversed or helped prevent inflammatory focal cardiac fibrosis, as well as glomerulosclerosis and focal renal fibrosis, induced by l-NAME. We considered that CCH prevented these disorders of the cardiovascular system by inhibiting ACE activity. Interestingly, in the thoracic aortas of the rats there were no histopathological changes induced by l-NAME given at 0.5 g/L/day. We therefore performed an additional experiment using a high concentration of l-NAME (1.0 g/L) for 8 weeks and then analyzed the histopathological changes in the thoracic aorta (Fig. 7). l-NAME (1.0 g/L)-induced hypertrophy of the arterial intima and the myofibrils was present of the thoracic aorta of rats receiving l-NAME alone. This damage was attenuated by CCH. Other researchers have reported the induction of vascular structural changes and hypertrophy and fibrosis of the myocardium by administration of l-NAME at 1.0 g/L.²⁷ One study reported that l-NAME caused coronary microvascular remodeling, but not remodeling of the large coronary vessels.²⁸ However, the present results suggested that CCH treatment inhibited the cardiovascular damage induced by l-NAME in the peripheral tissues, microvessels, and large vessels.

FIG. 7.

Histopathological photomicrographs of thoracic aorta sections from the control group, l-NAME group, and L + CCH group rats at 8 weeks post-treatment. Thoracic aorta sections were stained with hematoxylin and eosin (HE) and Masson's trichrome (MT) stains. Bar = 50 μm.

To investigate the antihypertensive mechanism of CCH and the pathway of prevention of cardiovascular remodeling in the heart, kidneys, and thoracic aorta, we determined the changes of NO_x concentration in sera of the L + CCH group rats. The serum concentration of NO_x did not change in rats that ingested CCH for 8 weeks. However, an increase in tissue ACE activity precedes the development of vascular and myocardial remodeling induced by long-term inhibition of NO synthesis.¹⁵ Therefore, we performed an additional experiment in which we gave a single dose of CCH to rats and measured the change in serum NO_x concentration. The NO level increased at 1 hour post-treatment of CCH and then returned to the basal level (Fig. 5). This result indicated that the serum NO level was transiently increased by treatment with CCH, suggesting that CCH treatment could ameliorate the l-NAME-induced inflammatory processes of hypertensive and cardiovascular damage in association with NO, an endothelium-derived relaxing factor. NO could inhibit the expression of cell adhesion molecules in the vascular endothelium.²⁹ This inhibition of adhesion molecules by NO may represent one of its important anti-inflammatory and anti-atherosclerotic actions. Recently, it has been demonstrated that ICAM-1 links leukocyte–endothelial cell interactions and changes in solute permeability.³⁰ Moreover, a soluble form of ICAM-1 has been detected in human serum and may be useful in inflammatory or autoimmune disorders.³¹ In the present study, increased levels of sICAM-1 in rats with l-NAME-induced inflammation were attenuated by treatment with CCH. Together with the data presented previously, we deduced a possible pathway for the protective effect of CCH treatment on attenuating hypertension and cardiovascular damage: first, CCH treatment resulted in an incremental increase of ACE inhibitory activity, which up-regulated the synthesis of NO gently. Second, the NO triggered the decrease of ICAM-1 concentration in inflamed tissues and microvessels, which led to low solute permeability of endothelial cells and links leukocyte–endothelial cell interactions. Then, cardiovascular damage was attenuated, and the hypertension and survival rate were improved.

Various studies have reported that several peptides derived from food materials reduce vascular resistance and have blood pressure-lowering effects in rats.³² We isolated nine specific peptides from human blood after oral intake of CCH, and we found that these peptides possessed ACE inhibitory activities,³³ although the mechanisms by which CCH inhibits remodeling activity have not been completely proven in vivo. More studies are needed to reveal the active substances in CCH and to clarify the antihypertensive and vasodilatory mechanisms of these substances.

Footnotes

Acknowledgment

This study was supported by a 2006 Grant-in-Aid for scientific research on “Study of the high value of foods and materials from stockbreeding that prevent lifestyle-related diseases,” from the Bio-oriented Technology Research Advanced Institution of Japan.

Author Disclosure Statement

All authors are employees of Nippon Meat Packers Inc. No competing financial interests exist.

References

Stamler

. Metabolic and nutritional factors in hypertension. Blood pressure and high blood pressure: aspects of risk. Hypertension, 1991; 18,Suppl I:95–107.

Yokoyama

, Chiba

, Yoshikawa

. Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried bonito. Biosci Biotechnol Biochem, 1992; 56:1541–1545.

Matsui

, Matsufuji

, Seki

, Osajima

, Nakashima

, Osajima

. Inhibition of angiotensin I-converting enzyme by Bacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle. Biosci Biotechnol Biochem, 1993; 57:922–925.

Kohama

, Matsumoto

, Oka

, Teramoto

, Okabe

, Mimura

. Isolation of angiotensin-converting enzyme inhibitor from tuna muscle. Biochem Biophys Res Commun, 1988; 155:332–337.

Sato

, Hosokawa

, Yamaguchi

et al. Angiotensin I-converting enzyme inhibitory peptides derived from wakame (Undaria pinnatifida) and their antihypertensive effect in spontaneously hypertensive rats. J Agric Food Chem, 2002; 50:6245–6252.

Cha

, Park

. Production and characterization of a soy protein-derived angiotensin I-converting enzyme inhibitory hydrolysate. J Med Food, 2005; 8:305–310.

Fuke

, Konosu

. Taste-active components in some foods: a review of Japanese research. Physiol Behav, 1991; 49:863–868.

Yao

, Zhang

, Zhou

. Effects of Ejiao (colla corii asini) on the hemodynamics, hemorheology and microcirculation during endotoxin shock in dogs [in Chinese] Zhongguo Zhong Yao Za Zhi, 1989; 14:44–46, 64.

Saiga

, Okumura

, Makihara

et al. Angiotensin I-converting enzyme inhibitory peptides in a hydrolyzed chicken breast muscle extract. J Agric Food Chem, 2003; 51:1741–1745.

10.

Saiga

, Iwai

, Hayakawa

et al. Angiotensin I-converting enzyme-inhibitory peptides obtained from chicken collagen hydrolysate. J Agric Food Chem, 2008; 56:9586–9591.

11.

Saiga

, Okumura

, Makihara

, Katsuda

, Morimatsu

, Nishimura

. Action mechanism of an angiotensin I-converting enzyme inhibitory peptide derived from chicken breast muscle. J Agric Food Chem, 2006; 54:942–945.

12.

Saiga-Egusa

, Iwai

, Hayakawa

, Takahata

, Morimatsu

. Antihypertensive effects and endothelial progenitor cell activation by intake of chicken collagen hydrolysate in pre- and mild-hypertension. Biosci Biotechnol Biochem, 2009; 73:422–424.

13.

Fujita

, Usui

, Kurahashi

, Yoshikawa

. Isolation and characterization of ovokinin, a bradykinin B1 agonist peptide derived from ovalbumin. Peptides, 1995; 16:785–790.

14.

Takemoto

, Egashira

, Tomita

et al. Chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade: effects on cardiovascular remodeling in rats induced by the long-term blockade of nitric oxide synthesis. Hypertension, 1997; 30:1621–1627.

15.

Takemoto

, Egashira

, Usui

et al. Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats. J Clin Invest, 1997; 99:278–287.

16.

Baylis

, Mitruka

, Deng

. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest, 1992; 90:278–281.

17.

Ribeiro

, Antunes

, de Nucci

, Lovisolo

, Zatz

. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension, 1992; 20:298–303.

18.

Cohen

. The role of nitric oxide and other endothelium-derived vasoactive substances in vascular disease. Prog Cardiovasc Dis, 1995; 38:105–128.

19.

Loscalzo

, Welch

. Nitric oxide and its role in the cardiovascular system. Prog Cardiovasc Dis, 1995; 38:87–104.

20.

Khan

, Harrison

, Olbrych

, Alexander

, Medford

. Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells. Proc Natl Acad Sci U S A, 1996; 93:9114–9119.

21.

Entman

, Youker

, Shappell

et al. Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism. J Clin Invest, 1990; 85:1497–1506.

22.

Chen

, Ye

, Li

, Yang

, Qian

, Hu

. Endothelium-independent vasorelaxant effect of sodium ferulate on rat thoracic aorta. Life Sci, 2009; 84:81–88.

23.

Pigott

, Dillon

, Hemingway

, Gearing

. Soluble forms of E-selectin, ICAM-1 and VCAM-1 are present in the supernatants of cytokine activated cultured endothelial cells. Biochem Biophys Res Commun, 1992; 187:584–589.

24.

Ohta

, Araki

, Shibata

et al. A novel in vivo assay system for consecutive measurement of brain nitric oxide production combined with the microdialysis technique. Neurosci Lett, 1994; 176:165–168.

25.

Kataoka

, Egashira

, Ishibashi

et al. Novel anti-inflammatory actions of amlodipine in a rat model of arteriosclerosis induced by long-term inhibition of nitric oxide synthesis. Am J Physiol Heart Circ Physiol, 2004; 286:H768–H774.

26.

, Manning RD

, Brands

. Long-term cardiovascular role of nitric oxide in conscious rats. Hypertension, 1994; 23:185–194.

27.

Michel

, Xu

, Blot

, Philippe

, Chatellier

. Improved survival in rats administered N^G-nitro l-arginine methyl ester due to converting enzyme inhibition. J Cardiovasc Pharmacol, 1996; 28:142–148.

28.

Numaguchi

, Egashira

, Takemoto

et al. Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats. Hypertension, 1995; 26:957–962.

29.

De Caterina

, Libby

, Peng

et al. Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest, 1995; 96:60–68.

30.

Sumagin

, Lomakina

, Sarelius

. Leukocyte-endothelial cell interactions are linked to vascular permeability via ICAM-1-mediated signaling. Am J Physiol Heart Circ Physiol, 2008; 295:H969–H977.

31.

Rothlein

, Mainolfi

, Czajkowski

, Marlin

. A form of circulating ICAM-1 in human serum. J Immunol, 1991; 147:3788–3793.

32.

Miguel

, Manso

, Aleixandre

, Alonso

, Salaices

, Lopez-Fandino

. Vascular effects, angiotensin I-converting enzyme (ACE)-inhibitory activity, and antihypertensive properties of peptides derived from egg white. J Agric Food Chem, 2007; 55:10615–10621.

33.

Iwai

, Zhang

, Kouguchi

, Saiga

, Shimizu

, Ohmori

. Blood concentration of food-derived peptides after oral intake of chicken collagen hydrolysate and its angiotensin-converting enzyme inhibitory activity of healthy volunteers. Food Sci Technol Res, 2009; 56:326–330.

Chicken Collagen Hydrolysate Protects Rats from Hypertension and Cardiovascular Damage

Abstract

Introduction

Materials and Methods

CCH preparation

Experimental animals

Measurement of systolic blood pressure (SBP)

Vasorelaxation assay

Determination of soluble ICAM-1 (sICAM-1) concentration

Determination of serum NOx concentration

Histopathological observations

Statistical evaluations

Results

Survival curves

Effect on antihypertensive measures

Effect on vasorelaxant measures

Effects on plasma sICAM-1 concentration

Changes in serum NOx concentration

Histopathology of heart, kidney, and thoracic aorta

Discussion

Footnotes

Acknowledgment

Author Disclosure Statement

References

Determination of serum NO_x concentration

Changes in serum NO_x concentration